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Seeing How Cas9 Got Its Groove Back, in 3D

The crystal structure of SpyCas9 features a nuclease domain lobe (red) and an alpha-helical lobe (gray) each with a nucleic acid binding cleft that becomes functionalized when Cas9 binds to guide RNA. [Lawrence Berkeley National Laboratory]

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Scientists are keen to realize the genome-editing potential of Cas9 enzymes to the fullest. While they have achieved some early successes, the scientists have also been hindered. They have lacked an understanding of the structural basis for Cas9’s functionality. No longer. Structural information is now available that illuminates not only the function, but also the regulation and evolution of the Cas9 enzyme family. With this information, scientists can look forward to the rational design of new and improved versions of Cas9 enzymes for basic research and genetic engineering.

The structural information emerged from research led by biochemist Jennifer Doudna and biophysicist Eva Nogales, both of whom hold appointments with the Lawrence Berkeley National Laboratory, the University of California, Berkeley, and Howard Hughes Medical Institute. The research team used X-ray crystallography to produce 2.6 and 2.2 angstrom resolution crystal structure images of two major Cas9 proteins—one from Streptococcus pyogenes (SpyCas) and Actinomyces naelslundii (AnaCas9). Then the team used single-particle electron microscopy to reveal how Cas9 partners with its guide RNA to interact with target DNA.

“The combination of x-ray protein crystallography and electron microscopy single-particle analysis showed us something that was not anticipated,” said Nogales. “The Cas9 protein, on its own, exists in an inactive state, but upon binding to the guide RNA, the Cas9 protein undergoes a radical change in its three-dimensional structure that enables it to engage with the target DNA.”

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Upon binding with guide RNA, the two structural lobes of Cas9 reorient so that the two nucleic acid binding clefts face each other, forming a central channel that interfaces with target DNA. [Lawrence Berkeley National Laboratory]

The researchers’ findings were published February 6 in Science, in an article entitled “Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.” In this article, the authors observed that the “architectures of Cas9 enzymes define nucleic acid binding clefts, and the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound.”

Despite significant differences outside of their catalytic domains, all members of the Cas9 family share the same structural core. This core is shaped rather like a clam. Instead of paired shells, the core has its two cleft-harboring lobes—a nuclease domain lobe and an alpha-helical lobe. Upon binding with guide RNA, the two lobes reorient so that the two clefts face each other, forming a central channel that interfaces with target DNA.

The authors emphasized that the identification of variable regions appended to a conserved Cas9 structural core provides a rationale for the diversity of crRNA:tracrRNA guide structures recognized by Cas9 enzymes. Also, these details suggest a framework for protein engineering approaches aimed at altering catalytic function, guide RNA specificity, or protospacer adjacent motif (PAM) requirements in targets.

Clarifying the point, Doudna said, “We see that the two structures are quite different from each other outside of their catalytic domains, suggesting an interesting structural plasticity that could explain how Cas9 is able to use different kinds of guide RNAs. Also, the differences in the two structures suggest that it may be possible to engineer smaller Cas9 variants and still retain function, an important goal for some genome engineering applications.”

The Cas9 results are of particular relevance to Type II CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems. These systems, which use Cas9 to generate double-strand breaks in DNA, are part of an adaptive bacterial immune response. They have also been harnessed as a gene editing tool in many eukaryotic organisms.

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